Adjuvants for use in vaccines

ABSTRACT

The invention relates to adjuvants that contain a lecithin, an oil and an amphiphilic surfactant and that are capable of forming a stable oil-in-water emulsion vaccine so as to minimize local reactions to the vaccine in the injected animal.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/117,705, filed Jan. 29, 1999 and U.S. ProvisionalApplication No. 60/121,760, filed Feb. 26, 1999.

FIELD OF THE INVENTION

[0002] The invention relates to immunological adjuvants. In particular,the invention relates to adjuvants which comprise an oil-in-wateremulsion and a surfactant. Adjuvants of the invention are useful in avariety of vaccine formulations, including vaccines comprising bacterialor viral components.

BACKGROUND OF THE INVENTION

[0003] The generation of immunity to infectious organisms is a powerfultool in disease control. Those antigens that induce immunity toinfection are known as immunogens. The protective antibody they inducemay collaborate with other natural defenses to inhibit the infectiveprocess, or they may neutralize harmful products of the infectiveorganism such as toxins.

[0004] An effective means of enhancing the antibody response is the useof an adjuvant. Thus, an adjuvant is included in a vaccine as anadditive or vehicle to enhance the response to the antigen. An adjuvantmay function by different mechanisms, including (1) trapping the antigenin the body to cause a slow release, (2) attracting cells of the immunesystem to the injection site, (3) stimulating cells of the immune systemto proliferate and to become activated, and (4) improving antigendispersion in the recipient's body.

[0005] A number of agents with diverse chemical properties have beenused as adjuvants, including water-in-oil and oil-in-water emulsions,mineral salts, polynucleotides and natural substances. One adjuvant,known under the trademark AMPHIGEN™, is described in U.S. Pat. No.5,084,269. AMPHIGEN™ adjuvant consists of de-oiled lecithin dissolved inan oil, usually light liquid paraffin. In vaccine preparations AMPHIGEN™is dispersed in an aqueous solution or suspension of the immunizingantigen as an oil-in-water emulsion.

[0006] Problems were observed when using an AMPHIGEN™ adjuvant accordingto U.S. Pat. No. 5,084,269, above. For example, the lecithin in theAMPHIGEN™ does not suffice to produce a stable emulsion of the oil, thusleading to a pool or depot of oil in the injected tissues. Mineral oilcan not be metabolized or removed by the animal. As a result, the oilbecomes a source of severe chronic inflammation and scarring.Emulsifying the AMPHIGEN™ directly in the antigenic preparation carriesthe risk of damaging the antigen. Also, if the desired emulsion fails toform, the valuable antigen must be discarded.

[0007] An adjuvant useful in vaccines for animals, including humans,that is effective and solves the above problems would therefore behighly desirable.

SUMMARY OF THE INVENTION

[0008] The invention relates to an adjuvant useful for the enhancementof the immune response of an animal to an antigen. In particular, theinvention relates to an adjuvant that is capable of forming anoil-in-water emulsion in a vaccine composition. The invention alsorelates to an adjuvant that, when used in a vaccine formulation, causesminimal inflammation and scarring at the vaccination site. The inventionfurther relates to a vaccine formulation that contains an adjuvant ofthe invention. Finally, the invention relates to a method of using anadjuvant of the invention in a vaccination.

[0009] In one embodiment, the adjuvant of the invention comprises alecithin, an oil and an amphiphilic surfactant capable of emulsifyingthe adjuvant, for example, a Tween or a Span surfactant. In anotherpreferred aspect, the surfactant is Tween 80, Tween 85, Span 80 or Span85.

[0010] In another embodiment, the adjuvant of the invention comprises alecithin, an oil and two amphiphilic surfactants capable of emulsifyingthe adjuvant or a vaccine composition that contains the adjuvant. In apreferred aspect, one of the two surfactants is predominantly found inthe aqueous phase, for example, Tween 80, and the other surfactant ispredominantly found in the oil phase, for example, Span 80.

[0011] A lecithin is a phosphatide. Crude preparations of lecithin mayinclude triglycerides. For purposes of the present invention, “lecithin”encompasses both purified and crude preparations. In a preferred aspect,the lecithin is de-oiled.

[0012] Suitable oils include a mineral oil, for example, DRAKEOL™ lightmineral oil.

[0013] In a further embodiment, the adjuvant of the invention containsan aqueous carrier solution, for example, a physiologically acceptablebuffer, water or a saline solution.

[0014] In a preferred embodiment, the adjuvant of the invention containsa lecithin, a mineral oil, two amphiphilic surfactants and an aqueouscarrier solution (e.g., saline).

[0015] In another embodiment of the invention, a method to inactivate aculture of Bordetella bronchiseptica (“B. bronchiseptica”) usingformalin and glutaraldehyde is described. In another aspect, a cultureof B. bronchiseptica is provided that was inactivated using formalin andglutaraldehyde. In yet another aspect, an antigen composition from a B.bronchiseptica culture is provided that was inactivated using formalinand glutaraldehyde. In yet another aspect, a vaccine composition isprovided that contains an antigen composition from a B. bronchisepticaculture that was inactivated using formalin and glutaraldehyde.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 presents a graph depicting the distribution of dropletsizes of an emulsion prepared as described below. Liries (a) and (b)depict that about 94% of the droplets have a diameter of 1 μm or less.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The invention relates to an adjuvant useful for the enhancementof the immune response to an antigen. In particular, the inventionrelates to an oily adjuvant that is capable of emulsifying a vaccineformulation. Further, the invention relates to an adjuvant that, whenused in a vaccine formulation, is capable of substantially avoiding theinflammation or scarring at the injection site, typical of vaccinescontaining mineral oil. Adjuvants of the invention comprise a lecithin,an oil and an amphiphilic surfactant capable of emulsifying the adjuvantor a vaccine composition that contains the adjuvant.

[0018] The invention is based, in part, on the discovery that addingfrom about 1.5% v/v (i.e., 1.5% volume per volume concentration obtainedby, e.g., mixing 98.5 volumes of the vaccine comprising the adjuvantwith 1.5 volumes of the amphiphilic surfactant) to 3.5% v/v of anamphiphilic surfactant to a vaccine containing an adjuvant as describedin U.S. Pat. No. 5,084,269 is effective to sufficiently emulsify avaccine composition formulated with such an adjuvant and to minimizeirritation in the injection site of the vaccinated animal.

[0019] In one embodiment, the adjuvant of the invention contains alecithin and an oil and an amphiphilic surfactant. In one embodiment,the adjuvant of the invention contains a lecithin and an oil and anamphiphilic surfactant capable of emulsifying a vaccine compositionformulated with an adjuvant of the invention. In another preferredembodiment, two amphiphilic surfactants are used in an adjuvant of theinvention, for example a Tween and a Span surfactant.

[0020] A preferred adjuvant, herein referred to as “No. 1 Adjuvant”,comprises about 2% v/v lecithin, about 18% v/v mineral oil, and about 8%v/v surfactant (e.g., about 5.6% v/v Tween 80 and about 2.4% v/v Span80), with the remaining volume being a saline solution. In a preferredaspect, a vaccine composition is formulated comprising an antigen at aconcentration of about 75% v/v and an adjuvant, preferably No. 1Adjuvant, at a concentration of about 25% v/v of the vaccinecomposition. All concentrations provided herein in percentage areindicated in volume per volume unless the context indicates otherwise.

Surfactants Useful in the Adiuvant of the Invention

[0021] Surfactants useful for the adjuvant of the invention areamphiphilic and acceptable for veterinary or medical use. Whether or nota particular surfactant is acceptable for medical or veterinary use canbe determined by those of ordinary skill in the art. A surfactant isamphiphilic if a part of the surfactant molecule is hydrophobic and apart is hydrophilic. See U.S. Pat. Nos. 5,690,942; 5,376,369; 4,933,179and 4,606,918, which describe surfactants than can be used in theadjuvant of the invention. Examples of surfactants useful in theadjuvant of the invention include, but are not limited to, a Tweensurfactant and a Span surfactant. Tween and Span surfactants include,but are not limited to, monolaureate (Tween 20, Tween 21, Span 20),monopalmitate (Tween 40, Span 40), monostearate (Tween 60, Tween 61,Span 60), tristearate (Tween 65, Span 65), monooleate (Tween 80, Tween81, Span 80) and trioleate (Tween 85, Span 85). In a preferredembodiment, Tween 80, Tween 85, Span 80 or Span 85 is used.

[0022] It is preferred that a surfactant useful in the adjuvant of theinvention is amphiphilic and has a hydrophilic-lipophilic balance(“HLB”) value that is preferably at least about half the sum of the HLBvalues of all other components of the adjuvant. More preferably, thesurfactant has an HLB value that is from about half to about twice thesum of the HLB values of all other components of the adjuvant. Morepreferably, the surfactant has an HLB value that is about the same asthe HLB value of all other components of the adjuvant. HLB values arereadily available for surfactants, lecithins, oils and carrier solutionsor, if necessary, can be determined through routine experimentation. Forexample, see U.S. Pat. Nos. 4,504,275 and 4,261,925 and referencesprovided therein.

[0023] Amphiphilic surfactants useful in the adjuvant of the inventionhave HLB values from about 2 to about 20, preferably from about 3 toabout 17. Methods for determinig the HLB value of particular surfactantsare known in the art. See for example U.S. Pat. Nos. 5,603,951;4,933,179 and 4,606,918, which describe surfactants having particularHLB values.

[0024] The concentration of a surfactant in a vaccine compositionformulated with the adjuvant of the invention is from about 1.5% to 3.5%v/v, more preferably from about 1.5% to about 3% v/v, more preferablyfrom about 1.5% to about 2.5%, and most preferably about 2% v/v. Whenmore than one surfactant is used, the sum of the concentrations of allsurfactants used in a vaccine composition formulated with the adjuvantof the invention is also from about 1.5% to 3.5%, more preferably fromabout 1.5% to about 3%, more preferably from about 1.5% to about 2.5%,and most preferably about 2% v/v.

[0025] The concentration of a surfactant in the adjuvant of theinvention also depends on the concentration at which the adjuvant isused in a vaccine composition. For example, a vaccine composition may beformulated with the adjuvant of the invention so that about 25% of thevolume of the vaccine composition is the adjuvant (“25% adjuvant”) andthe remaining about 75% is made up of other components, for example theantigen composition. In one aspect, the concentration of the surfactantin a 25% adjuvant is from about 6% to 14% v/v. More preferably, thesurfactant concentration in a 25% adjuvant is from about 6% to about12%, more preferably from about 6% to about 10%, and most preferablyabout 8% v/v.

[0026] The concentration of the surfactant in the adjuvant of theinvention is dependent on different factors. For example, the higher theconcentration of oil in the adjuvant the more surfactant is required toemulsify a vaccine composition formulated with the adjuvant of theinvention. Another factor that is useful to determine the concentrationof a surfactant is the concentration of a lecithin. The higher theconcentration of a lecithin in the adjuvant, the less surfactant may berequired for emulsification.

[0027] When the adjuvant of the invention is used in a vaccinecomposition at a concentration of less than 25% v/v, the concentrationof the adjuvant components in the adjuvant has to be increasedaccordingly. The aqueous carrier is an exception as the carrier alwayscomprises the volume that remains unoccupied by all other components;thus if the concentration of all components except the carrierincreases, the concentration of the carrier in the adjuvant willdecrease and vice versa. For example, when the adjuvant is used at aconcentration of about 12.5% v/v in a vaccine composition, theconcentration of the components in the adjuvant is about twice theconcentration of the components in a 25% adjuvant. Similarly, when theadjuvant of the invention is used in a vaccine composition at aconcentration that is above 25% v/v, the concentration of the componentsin the adjuvant has to be decreased accordingly, for example when theadjuvant is used at a concentration of about 50% v/v in a vaccinecomposition, the concentration of the components in the adjuvant isabout half the concentration of the components in a 25% adjuvant.

[0028] In one embodiment, two amphiphilic surfactants may be used in theadjuvant of the invention. Preferably, the two surfactants would includeone surfactant that would be more concentrated in an aqueous phase thanin an oil phase of the adjuvant (“hydrophilic surfactant”) and onesurfactant that would be more concentrated in an oil phase of theadjuvant (“lipophilic surfactant”). For example, Tween 80 wouldconcentrate more in an aqueous phase and Span 80 would concentrate morein an oil phase. A preferred hydrophilic surfactant has an HLB valuefrom about 9 to about 20 and a preferred lipophilic surfactant has anHLB value from about 2 to about 9. See U.S. Pat. Nos. 5,603,951;4,933,179 and 4,606,918, which describe surfactants with HLB values inboth ranges useful for the adjuvant of the invention.

[0029] When two surfactants are used in the adjuvant of the invention,the total concentration of both surfactants combined in a vaccinecomposition formulated with the adjuvant of the invention is from about1.5% to 3.5%, more preferably from about 1.5% to about 3%, morepreferably from about 1.5% to about 2.5%, and most preferably about 2%v/v. The concentration of each of two surfactants used in the adjuvantof the invention may differ from each other. For example, when ahydrophilic surfactant and a lipophilic surfactant are used, for exampleTween 80 and Span 80, the concentration of Tween 80 may be from about1.2× to about 5×, more preferably from about 1.5× to about 4×, morepreferably from about 1.8× to about 3×, more preferably from about 2× toabout 2.5× and more preferably about 2.3× as high as the concentrationof Span 80, preferably when used in an adjuvant with a lecithin and anoil concentration as in No. 1 Adjuvant.

[0030] The concentration of the hydrophilic surfactant used in theadjuvant of the invention depends, in part, on the size of the aqueousphase, and the concentration of the lipophilic surfactant depends, inpart, on the size of the oil phase. In one embodiment, the adjuvant ofthe invention that consists of an aqueous phase at 80% v/v and of an oilphase at 20% v/v, may contain a hydrophilic surfactant at aconcentration of up to about 4 times (i.e., 80/20) the concentration ofa lipophilic surfactant, or for example up to about 2 times.

Non-Surfactant Components of the Adjuvant of the Invention

[0031] In addition to an amphiphilic surfactant, the adjuvant of theinvention contains a lecithin and an oil. In another aspect, theadjuvant of the invention contains an aqueous carrier solution.

[0032] Any lecithin known in the art is useful for the adjuvant of theinvention. Lecithin refers to a mixture of phosphatides. When providedas a crude extract, a lecithin may also contain triglycerides. Lecithinsmay be of plant or animal origin. In addition, lecithins may besynthetically derived. Examples of lecithins are described in U.S. Pat.Nos. 5,690,942; 5,597,602 and 5,084,269. In a preferred embodiment, thecontents of triglycerides in a lecithin used in the adjuvant of theinvention is lowered compared to its natural source, i.e., the lecithinis de-oiled. A number of ways are known in the art to de-oil a lecithin,for example as described in U.S. Pat. No. 5,597,602.

[0033] The concentration of a lecithin in a vaccine compositionformulated with the adjuvant of the invention is from about 0.25% toabout 12.5% v/v, more preferably from about 0.5% to about 10% v/v, morepreferably from about 0.5% to about 7.5%, more preferably from about0.5% to about 5%, more preferably from about 0.5% to about 2.5%, andmost preferably from about 0.5% to about 1.25% v/v.

[0034] The concentration of a lecithin in a 25% adjuvant is at leastabout 1% v/v, preferably at least about 2% v/v. In another aspect, thelecithin concentration in a 25% adjuvant is from about 1% to about 50%v/v, more preferably from about 2% to about 40% v/v, more preferablyfrom about 2% to about 30% v/v, more preferably from about 2% to about20% v/v, more preferably from about 2% to about 10% v/v and mostpreferably from about 2% to about 5% v/v. The concentration of alecithin in the adjuvant of the invention with a higher or lowerconcentration is determined as exemplified above.

[0035] The adjuvant of the invention contains an oil, for example an oildescribed in U.S. Pat. Nos. 5,814,321; 5,084,269. In a preferred aspect,the adjuvant of the invention contains a mineral oil, for exampleDRAKEOL™. In another aspect, a mixture of oils is used. The oil may beprovided for preparation of the adjuvant of the invention as pure oil oras a mixture that contains the oil and another component, for examplethe lecithin.

[0036] The concentration of an oil in a vaccine composition formulatedwith the adjuvant of the invention is from about 1% to about 23% v/v,more preferably from about 1.5% to about 20% v/v, more preferably fromabout 2.5% to about 15%, more preferably from about 3.5% to about 10%,more preferably from about 3.5% to about 7.5%, more preferably fromabout 4% to about 6% v/v, and most preferably about 4.5%.

[0037] The concentration of an oil in a 25% adjuvant is at least about5% v/v, preferably at least about 8% v/v and more preferably at leastabout 12% v/v. In another aspect, the oil concentration in a 25%adjuvant is from about 4% to about 92% v/v, more preferably from about6% to about 80% v/v, more preferably from about 10% to about 60% v/v,more preferably from about 14% to about 40% v/v, more preferably fromabout 14% to about 30% v/v, more preferably from about 16% to about 24%and most preferably about 18%. The concentration of an oil in theadjuvant of the invention with a higher or lower concentration isdetermined as exemplified above.

[0038] In another embodiment, an aqueous carrier is used in the adjuvantof the invention, for example saline (e.g., phosphate-buffered saline),tris-HCl, citrate-phosphate buffer, Hepes buffers, otherpharmaceutically acceptable buffers known in the art or water. The pH ofthe carrier preferably is physiologically acceptable, for examplebetween 6 and 8, most preferably around 7. The aqueous carrier used inthe adjuvant of the invention preferably takes up the volume that is notneeded for any of the other components.

[0039] The adjuvant of the invention is preferably provided at aconcentration that is from about 2× to about 10× the concentration afterformulation of the adjuvant in a vaccine composition, more preferablyfrom about 2× to about 8×, more preferably from about 3× to about 6× andmost preferably about 4×.

Uses of Adjuvants of the Invention

[0040] Adjuvants of the invention may be used to enhance the immuneresponse to an antigen of a vaccine formulation. Adjuvants of theinvention can be used with antigens derived from any bacteria or fromany virus, provided the antigen does not get destroyed or denatured.Examples of antigens, and not by way of limitation, are Erysipelothrixrhusiopathiae antigens, Bordetella bronchiseptica antigens, antigens oftoxigenic strains of Pasteurella multocida, antigens of Eschericia colistrains that cause neonatal diarrhea, Actinobacillus pleuropneumoniaeantigens, Pasteurella haemolytica antigens, or any combination of theabove. Adjuvants of the invention are also useful in vaccinecompositions that contain an antigen described in U.S. Pat. Nos.5,616,328 and 5,084,269.

[0041] In a preferred embodiment, the adjuvant of the invention is usedin a vaccine formulation containing an antigen obtained from the liquidphase of an Erysipelothrix rhusiopathiae (“E. rhusiopathiae”) culture.In a preferred aspect, a culture of E. rhusiopathiae is inactivated byadding formalin (about 0.5% v/v final concentration) and, afterincubation for 24 hours at 37° C., the cells were removed, for exampleby centrifugation or filtration. The culture supernatant, in a preferredembodiment, is concentrated about 10 told and aluminum hydroxide gel(preferably REHYDRAGEL™) is added to the concentrated supernatant at afinal concentration of about 30% v/v to stabilize the antigen. Inanother preferred embodiment, thimerosal (about 0.01% v/v finalconcentration) (Dimportex, Spain, imported through Flavine Inc.,Klosters, N.J.) with EDTA (about 0.07% v/v final concentration) areadded to the antigens as preservatives. In another preferred embodiment,a vaccine composition is formulated comprising the antigen and theadjuvant of the invention (e.g. No. 1 Adjuvant) with the adjuvantcomprising, for example, about 25% v/v of the vaccine composition. Thispreferred E. rhusiopathiae antigen is described in U.S. PatentApplication Serial No. 60/117,704, filed Jan. 29, 1999, entitled“Erysipelothrix rhusiopathiae Antigens and Vaccine Compositions”, whichis incorporated herein by reference.

[0042] In another preferred embodiment, the adjuvant of the invention isused in a vaccine composition containing antigens from a B.bronchiseptica culture that has been inactivated by adding formalinthereto in log phase, preferably late log phase, followed by theaddition of glutaraldehyde. In addition to killing the bacterial cells,the purpose of this novel and unique inactivation is to make nontoxicthe endotoxin and exotoxin B. bronchiseptica, while leaving the antigensof B. bronchiseptica cells effective in eliciting the desired immuneresponse. Formalin is added to a concentration in the B. bronchisepticaculture of from about 0.2% v/v to about 1% v/v, more preferably fromabout 0.4% v/v to about 0.8% v/v and most preferably about 0.6% V/v.Glutaraldehyde is added from about 10 minutes to about 40 minutesfollowing the addition of formalin to the culture, more preferably fromabout 15 minutes to about 30 minutes and most preferably about 20minutes. Glutaraldehyde is added to a concentration in the B.bronchiseptica culture of from about 0.2% v/v to about 1% v/v, morepreferably from about 0.4% v/v to about 0.8% v/v and most preferablyabout 0.6% v/v. Prior to adding the glutaraldehyde to the culture, ithas a concentration of from about 10% v/v to about 50% v/v, morepreferably from about 15% v/v to about 35% v/v and most preferably about25% v/v. Following the addition of formalin and glutaraldehyde to the B.bronchiseptica culture, the resulting mix is incubated under stirring atfrom about 32° C. to about 42° C., more preferably at from about 35° C.to about 39° C. and most preferably at about 37° C. The mix is incubatedfrom about 12 hours to about 60 hours, more preferably from about 24hours to about 48 hours. All other processing steps in preparing anantigen composition of the invention from B. bronchiseptica culture aredescribed in Example 7, infra, and in U.S. Pat. Nos. 5,019,388 and4,888,169.

Vaccine Compositions Comprising Adjuvants of the Invention and theirAdministration

[0043] The adjuvant of the invention may be used in a vaccineformulation to immunize an animal. In one embodiment, the vaccineformulation contains the adjuvant of the invention and an antigen. Theoptimal ratios of each component in the vaccine formulation may bedetermined by techniques well known to those skilled in the art.

[0044] A vaccine formulation may be administered to a subject per se orin the form of a pharmaceutical or therapeutic composition.Pharmaceutical compositions comprising the adjuvant of the invention andan antigen may be manufactured by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes. Pharmaceuticalcompositions may be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients or auxiliarieswhich facilitate processing of the antigens of the invention intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen. For purposes of thisapplication, “physiologically acceptable carrier” encompasses carriersthat are acceptable for human or animal use without relatively harmfulside effects (relative to the condition being treated), as well asdiluents, excipients or auxiliaries that are likewise acceptable.

[0045] Systemic formulations include those designed for administrationby injection, e.g. subcutaneous, intradermal, intramuscular orintraperitoneal injection.

[0046] For injection, the vaccine preparations may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks's solution, Ringer's solution, phosphate buffered saline, orany other physiological saline buffer. The solution may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the proteins may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

[0047] Determination of an effective amount of the vaccine formulationfor administration is well within the capabilities of those skilled inthe art, especially in light of the detailed disclosure provided herein.

[0048] An effective dose can be estimated initially from in vitroassays. For example, a dose can be formulated in animal models toachieve an induction of an immune response using techniques that arewell known in the art. One having ordinary skill in the art couldreadily optimize administration to all animal species based on resultsdescribed herein. Dosage amount and interval may be adjustedindividually. For example, when used as a vaccine, the vaccineformulations of the invention may be administered in about 1 to 3 dosesfor a 1-36 week period. Preferably, 1 or 2 doses are administered, atintervals of about 3 weeks to about 4 months, and booster vaccinationsmay be given periodically thereafter. Alternative protocols may beappropriate for individual animals. A suitable dose is an amount of thevaccine formulation that, when administered as described above, iscapable of raising an immune response in an immunized animal sufficientto protect the animal from an infection for at least 4 to 12 months. Ingeneral, the amount of the antigen present in a dose ranges from aboutlpg to about 100 mg per kg of host, typically from about 10 pg to about1 mg, and preferably from about 100 pg to about 1 pg. Suitable doserange will vary with the route of injection and the size of the patient,but will typically range from about 0.1 mL to about 5 mL.

[0049] The invention having been described, the following examples areoffered by way of illustration and not limitation.

EXAMPLE 1 The Use of an Adiuvant that Contains Oil and Lecithin

[0050] The following example describes the use of an adjuvant thatcontains de-oiled lecithin dissolved in an oil (“oil-lecithinadjuvant”), usually mineral oil (light liquid paraffin) in veterinaryvaccines. See U.S. Pat. No. 5,084,269, which describes an oil-lecithinadjuvant. A vaccine preparation using an oil-lecithin adjuvant is anoil-in-water emulsion.

[0051] All percentage concentrations herein are provided in volume pervolume unless indicated otherwise. Percentage values, unless otherwiseindicated, of an oil-lecithin adjuvant refer to the concentration of amixture of lecithin (10% of the mixture) and a carrier oil (DRAKEOL™)(90% of the mixture) in an aqueous carrier (continuous phase). Forexample, a 20% oil-lecithin adjuvant contains 2% v/v lecithin (CentralSoya, Fort Wayne, Ind.), 18% v/v DRAKEOL™. 5 (Penreco, Karns City, Pa.)and 80% v/v saline solution (with the saline content being reduced ifother components, for example surfactants, are added). The percentagevalues of an oil-lecithin adjuvant in a vaccine composition, i.e.,following dilution of the adjuvant solution with the antigen solution,refer to the concentration of a mixture of lecithin (10% of mixture) anda carrier oil (DRAKEOL™) (90% of mixture) in the vaccine preparationwhich comprises the adjuvant and a solution containing an antigen,unless the context indicates otherwise. In all cases where a surfactantwas added to an adjuvant composition, the percentage values for asurfactant concentration refer to the total concentration of all addedsurfactants in the adjuvant or the vaccine preparation, unless thecontext indicates otherwise.

[0052] When an oil-lecithin adjuvant was used as an adjuvant in vaccineformulations, it was found that it does not emulsify aqueouspreparations without the addition of extra surfactants as the lecithinin the oil-lecithin adjuvant did not suffice for emulsification.Therefore, vaccines made using inadequately dispersed oil-lecithinadjuvant formed a pool or depot of mostly mineral oil in the tissues atthe injection site. This oil can not be metabolized or removed by theinjected animal and so it remains as a source of severe chronicinflammation and scarring.

[0053] It was also determined that adding surfactants to a vaccineformulation comprising an oil-lecithin adjuvant and an antigen in orderto emulsify the formulation was not an adequate solution. Problemsencountered when adding oil and surfactants to the vaccine formulationbefore emulsifying were that the antigen could get damaged and, if asuitable emulsion was not achieved, that the formulation would have tobe discarded including the valuable antigen.

[0054] Different adjuvant compositions were tested comprising anoil-lecithin adjuvant in combination with surfactants to emulsify thevaccine formulations.

EXAMPLE 2 The Use of an Adjuvant Containing a Surfactant at a LowConcentration

[0055] The following example describes the use of an emulsion containing40% oil-lecithin and 2% of synthetic surfactants, i.e., Tween 80 andSpan 80 (Van Water & Rogers, Omaha, Nebr.) in phosphate buffered saline.This adjuvant was prepared aseptically and separate from the antigen.The emulsion was added to the antigen preparation without furtheremulsification. The synthetic surfactants helped the oil-lecithinadjuvant to disperse as a coarse, relatively stable emulsion. Theadjuvant emulsion was added to the aqueous antigenic preparation at therate of one in eight, decreasing the oil-lecithin adjuvant content from40% to 5%, and the surfactants from a combined 2% to 0.25%.

[0056] The adjuvant was used in several vaccines. It was found thatbecause the emulsion is coarse and not very stable, the oil dropletstend to coalesce and to separate as a permanent, irritating depot of oilin the injected tissues. Another problem observed with this adjuvant wasthat it aggregates with Al gel. A number of vaccines contain Al gel fora number of purposes like, for example, as an adjuvant or to stabilizean antigen or to bind endotoxin. The oil-lecithin adjuvant carries anegative charge which causes it to bind to the positively charged Al gelto form coarse aggregates. These aggregates are unsightly, difficult topass through a hypodermic needle, and very irritating to the injectedtissues.

EXAMPLE 3 The Use of an Adjuvant Containing a Surfactant at a HighConcentration

[0057] An oil-lecithin adjuvant (5% v/v) was emulsified in the antigenicpreparation with the help of Tween 80 and Span 80 surfactants, as above,but at a total surfactant concentration of 8% in the vaccinecomposition. The emulsion was very fine and stable. It had almost theclarity of a solution and it did not cream on standing. Under themicroscope, with maximum magnification (resolution 0.2 micron), mostdroplets were too small to be visible. Thus, it was a microemulsion.This adjuvant, when used in a vaccine formulation, was found to bevirtually free of injection-site reactivity and, when Al gel was added,there was no detectable aggregation of oil and gel. As a result of itshigh surfactant content, this adjuvant is easy to emulsify, attractivein appearance, stable, unreactive with Al gel, and virtually free ofirritating effects at the site of vaccination. Despite these advantages,however, this emulsion had slightly lower adjuvant potency compared tothe coarse version made with surfactants at a low concentration.

EXAMPLE 4 The Use of an Adjuvant Containing a Surfactant at a MediumConcentration

[0058] An attempt was made to find an adjuvant emulsion that isacceptably smooth and fully potent as an adjuvant. A 20% oil-lecithinadjuvant was used in these experiments as it was found that a 20%oil-lecithin adjuvant emulsion is easier to make than a 40% oil-lecithinadjuvant emulsion. Its addition to vaccines at a rate of one in four, tomake a final oil concentration of 5%, would leave 75% of the dose volumefor antigens. Preliminary experiments showed that a smooth submicronemulsion (most droplets had a diameter of less than one micron, seeFIG. 1) could be prepared with 20% oil and 16% of Tween 80 and Span 80surfactants.

[0059] Two emulsions were prepared for the assays. One contained a 20%oil-lecithin adjuvant and 16% of Tween 80 and Span 80 surfactants.Diluting it one in four resulted in an emulsion comprising 5%oil-lecithin adjuvant and 4% surfactants in the vaccine preparation. Theother emulsion was prepared with a 40% oil-lecithin adjuvant and 2% ofTween 80 and Span 80 surfactants. Diluting it one in eight gave anemulsion with 5% oil-lecithin adjuvant and 0.25% surfactants.

[0060] Al gel (REHYDRAGEL™ obtained from Reheis, Berkeley Heights, N.J.)was added to a concentration of 10% to samples of each emulsion. In theemulsion with 0.25% surfactants the oil and Al gel aggregated andseparated to form a thick layer at the top of the liquid column(creaming). In the emulsion with 4% surfactants, by contrast, there wasno aggregation or creaming. With 4% surfactants, the Al gel sedimentedat the bottom of the tube leaving the oil droplets dispersed in thesupernatant fluid.

EXAMPLE 5 Swelling of Injection Sites when Using an Adjuvant Containinga Surfactant at a Medium Concentration

[0061] Vaccine preparations were tested in pigs to determine whetherswelling of the injection site occurred when an adjuvant with a mediumconcentration of surfactant was used. Vaccine preparations that containa 5% oil-lecithin adjuvant and either 0.25% or 4% surfactants caused noswelling in pigs at the injection site. When Al gel was added to thevaccine preparation at a concentration of 10%, the preparation with0.25% surfactants caused severe injection site swellings whereas the onewith 4% surfactants resulted in almost no swelling.

[0062] Experiments were carried out to determine the range of surfactantconcentrations that are effective in preventing aggregation with Al geland swelling of the injection site. When using a 1.5% surfactantconcentration in the vaccine, slight aggregation of oil and Al gel wasobserved. The aggregation was much heavier at lower surfactantconcentrations. At 2% and 4% surfactant concentrations, there was noaggregation. The swelling induced in pigs by vaccine preparationscontaining 0.5% or less surfactants were larger at 2 and 4 weeks aftervaccination than those induced by preparations with 1% or moresurfactants. By 6 weeks after vaccination, it was evident that 1.5%surfactants was the minimum needed to avoid chronic swellings.

EXAMPLE 6 Adjuvants with Useful In Vitro and In Vivo Properties

[0063] Assays were carried out to find an adjuvant that does not reactwith Al gel and does not lead to reactivity in the animal followingvaccination. A 20% oil-lecithin adjuvant that contains 8% surfactants,resulting in a vaccination preparation with 5% oil-lecithin adjuvant and2% surfactant, was determined to be sufficient to avoid both in vitroreactivity with Al gel and irritation of the tissues at the vaccinationsite. Evidence of a relationship between surfactant concentration andadjuvant power was much less clear. There were occasional indicationsthat 4% surfactants in the vaccine was excessive, e.g. in the inductionof agglutinin to E. coli K99, and neutralizing antitoxin to the toxin ofP. multocida Type D.

[0064] Thus, it was determined that the optimal concentration ofsurfactants was 8% in a 20% oil-lecithin adjuvant, resulting in 2%surfactants in the vaccine composition. This provided for reasonablyeasy emulsification and for good stability in cold storage. In vaccineswith 5% oil-lecithin adjuvant, 2% surfactants was ideal for bothadjuvant power and freedom from irritancy in the injected tissues.

[0065] The droplet size in the submicron emulsion of a 20% oil-lecithinadjuvant with 8% surfactants was determined. The 8% surfactant consistedof 5.6% Tween 80 in the aqueous phase and 2.4% Span 80 in the oil phase.About 94% of all droplets were less than 1 micron in diameter, see FIG.1.

[0066] A stock of 100 mL of a 20% oil-lecithin adjuvant with 8%surfactants was made from 200 mL filter-sterilized lecithin-oil solution(10% lecithin in DRAKEOL™ mineral oil), autoclaved Tween 80 (56 mL) andSpan 80 (24 mL), and phosphate buffered saline (720 mL) (Dulbecco PBS).The lecithin-oil solution and Span 80 were combined and mixed in asterile tank for at least 1 hour at room temperature untilemulsification was complete. The saline and Tween 80 were combined andmixed in a sterile tank for at least 1 hour at room temperature. The oilmixture was emulsified in the aqueous mixture using an emulsifier.Emulsification was continued by recirculation until all of the adjuvantwas added into the saline. The emulsion was then passed twice through ahomogenizer at room temperature. The adjuvant was stored at 2 to 80 C.

EXAMPLE 7 Atrophic Rhinitis Vaccine Using an Adjuvant Containing aSurfactant at a Medium Concentration

[0067] The adjuvant as described in Example 4 with a mediumconcentration of surfactants was used in an Atrophic Rhinitis Vaccinewhich contained antigens of Bordetella bronchiseptica and toxigenicPasteurella multocida. A Bordetella bronchiseptica-Pasteurella multocidaBacterin-Toxoid vaccine was made from B. bronchiseptica cells and thetoxoid of P. multocida.

[0068]B. bronchiseptica cells, strain 2-9 NADL, were prepared asdescribed in U.S. Pat. Nos. 5,019,388 and 4,888,169 except that at theend of the growth cycle, cultures were continuously mixed and formalinsolution was added to a final concentration of 0.6%. Within 20 minutesafter the addition of formalin, a 25% glutaraldehyde solution was addedto a final concentration of 0.6%. The culture was stirred for 24 to 48hours at 37+2° C. to complete inactivation and detoxification. (SeeTable 1). Then, the culture fluids were cooled to 15° C. or less forprocessing. Inactivated cultures not processed immediately were storedat 2 to 8° C. for up to 14 days. Following inactivation, the bacteriawere separated from the culture fluid by centrifugation. The supernatantwas discarded and the cells were resuspended in phosphate-bufferedsaline at approximately one tenth of the original volume. Theconcentrated suspension was stored at 2 to 8° C. The treatment of B.bronchiseptica with two aldehydes inactivates both the endotoxin and theexotoxin, obviating other treatments for safety.

[0069] The toxoid of P. multocida was prepared in two different forms asdescribed in U.S. Pat. Nos. 5,536,496 and 5,695,769. In one form, thetoxin is toxoided within the bacterial cells by the addition offormaldehyde to the culture; the toxoid stays inside the cells. In theother form, the live cells are mechanically disrupted and the toxinextracted. The toxin is toxoided by exposure to a high pH, as describedin U.S. Pat. No. 5,536,496. Both forms of the toxoid are treated with Algel to control free endotoxin by a patented process, as described inU.S. Pat. No. 5,616,328. (See Table 1). A synergy between the two formsof pasteurella toxoid results in antitoxin responses far exceeding thesum of the responses to each form when used alone as described in U.S.Pat. No. 5,695,769. TABLE 1 Treatment of cells during preparation of theBordetella Bronchiseptica-Pasteurella Multocida Bacterin-Toxoid vaccineTarget Temp. Time Antigen Inactivant Concentration Range (Min/Max) B.bronchiseptica Formalin 0.6% 37 ± 2° C. 24-48 hours Glutaraldehyde 0.6%P. multocida cell-bound Formalin 0.4% 37 ± 2° C. 5 days toxoid P.multocida cell-free 5N NaOH pH 10 ± 0.2 15 ± 2° C. 1 to 6 hours toxoid

[0070] The adjuvant described in Example 4 was added to result inconcentrations of 5% oil-lecithin adjuvant and 2% surfactants in thevaccine formulation.

[0071] A trial to determine the minimum immunizing dose of the atrophicrhinitis vaccine demonstrated the adjuvant properties of theoil-lecithin adjuvant with a medium concentration of surfactants.Pregnant sows were vaccinated with two 2 mL doses at an interval of 4weeks. They farrowed about 2 weeks after the second dose. At one monthof age, their pigs were subjected to a severe challenge, consisting ofvirulent B. Bronchiseptica and P. Multocida cultures given intranasallyin sequence. The pigs born to 7 sows vaccinated only with a placebodeveloped severe atrophic rhinitis. The litters of all 7 sows givenvaccine containing a full dose of the antigens were strongly protectedby the maternal antibody still in circulation. Sows given vaccinescontaining ½ or ⅛ dose of antigens did not provide their litters withsatisfactory protection.

EXAMPLE 8 ErysiDelas Vaccine Using an Adjuvant Containing a Surfactantat a Medium Concentration

[0072] The adjuvant described in Example 4 with a medium concentrationof surfactants was used in an erysipelas vaccine which contained E.rhusiopathiae antigens. The antigens for use in a vaccine were made fromE. rhusiopathiae cultures inactivated with 0.5% formalin for at least 24hours. The inactivated cultures were clarified by centrifuging, andconcentrated roughly tenfold by molecular filtration. The concentrateswere stabilized by adding Al gel, i.e., REHYDRAGEL™, to a concentrationof 30%. The preadsorbed concentrates were included in the vaccine in anamount such that each 2 mL dose contained at least 3.2 opacity units(OU) as calculated from the optical density (OD) of the culture atinactivation. (The OD is multiplied by the final concentration factor togive a value in OU per mL.)

[0073] An Erysipelas vaccination was carried out to determine theefficacy of a vaccine containing the oil-lecithin adjuvant with a mediumconcentration of surfactants. The adjuvant as described in Example 4 wasadded at a final concentration of 25% v/v, giving a final lecithin oilconcentration of 5%. Thimerosal (0.01% w/v), with EDTA (0.07% w/v), wasadded as preservative.

[0074] Vaccines made according to this formula were tested twice forefficacy in pigs. In each case pigs were vaccinated with two 2 mL dosesgiven intramuscularly (IM) one dose at about 3 weeks (weaning) and thesecond dose 3 weeks later. Controls received phosphate-buffered salineas a placebo. Immunity was challenged by the IM injection of virulent E.Rhusiopathiae at about 9 weeks of age in one study and 6 months of agein the other. As shown in Table 2, protection due to vaccination was100% at 9 weeks and 75% at 6 months (i.e., slaughter age). These resultsindicated that the vaccine provides satisfactory protection againstErysipelas throughout the normal feeding period.

[0075] The vaccine used in the group challenged at 9 weeks was already12 months old. The result confirms that the protective antigen wassuccessfully stabilized. TABLE 2 Protection of pigs against ErysipelasControls Vaccinates Age at Challenge (Protected/Challenged)(Protected/Challenged) 9 weeks 0/10 19/19 6 months 0/10 15/20

[0076] Note: In the vaccinated group challenged at 9 weeks, the 20th pigwas excluded. A very fractious animal, it struggled so violently whenhandled that its temperature at rest could not be determined. Followingchallenge this pig remained completely healthy.

[0077] The invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention, and any adjuvants which are functionally equivalent arewithin the scope of the invention. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

[0078] All publications cited herein are incorporated by reference intheir entirety.

What is claimed is:
 1. A vaccine composition comprising from about 0.25%to about 12.5% v/v of a lecithin, from about 1% to about 23% v/v of anoil, from about 1.5% to 3.5% v/v of at least one amphiphilic surfactantand an antigen.
 2. The vaccine composition of claim 1 wherein said oilis a mineral oil and wherein said lecithin is de-oiled.
 3. The vaccinecomposition of claim 1 further comprising an aqueous carrier.
 4. Thevaccine composition of claim 1 wherein said antigen is selected from thegroup consisting of Erysipelothrix rhusiopathiae antigens, Bordetellabronchiseptica antigens, Pasteurella multocida antigens, antigens ofEschericia coli strains that cause neonatal diarrhea, Actinobacilluspleuropneumoniae antigens, Pasteurella haemolytica antigens andcombinations thereof.
 5. The vaccine composition of claim 1 wherein twoamphiphilic surfactants are used.
 6. The vaccine composition of claim 5wherein one hydrophilic surfactant and one lipophilic surfactant areused.
 7. The vaccine composition of claim 5 wherein said lecithin is ata concentration of about 0.5% v/v, wherein said oil is at aconcentration of about 4.5% v/v and wherein said amphiphilic surfactantsare at a concentration of about 2% v/v.
 8. The vaccine composition ofclaim 5 further comprising an aqueous carrier.
 9. An adjuvantcomposition comprising from about 0.25% to about 12.5% v/v of alecithin, from about 1% to about 23% v/v of an oil and from about 1.5%to 3.5% v/v of at least one amphiphilic surfactant when said adjuvant isformulated in a vaccine composition.
 10. A method of making a vaccinecomposition comprising adding an adjuvant composition of claim 9 to anantigen composition.
 11. The method of claim 10 wherein said antigen isselected from the group consisting of Erysipelothrix rhusiopathiaeantigens, Bordetella bronchiseptica antigens, Pasteurella multocidaantigens, antigens of Eschericia coli strains that cause neonataldiarrhea, Actinobacillus pleuropneumoniae antigens, Pasteurellahaemolytica antigens and combinations thereof.
 12. An antigencomposition comprising a Bordetella bronchiseptica culture that has beeninactivated by adding formalin followed by adding glutaraldehyde.
 13. Avaccine composition comprising the antigen composition of claim 12 andan adjuvant.
 14. The vaccine composition of claim 13 wherein saidadjuvant is the adjuvant of claim
 1. 15. A method of inactivating aBordetella bronchiseptica culture comprising adding formalin to saidculture followed by adding glutaraldehyde to said culture.
 16. ABordetella bronchiseptica composition comprising a Bordetellabronchiseptica culture, formalin and glutaraldehyde.
 17. A vaccine forprotecting a mammal against a Bordetella bronchiseptica infectioncomprising an amount of Bordetella brochiseptica cells from a cultureinactivated according to the method of claim 15 effective to protectsaid mammal against a Bordetella bronchiseptica infection and aphysiologically acceptable carrier.
 18. A vaccine according to claim 17,further comprising an amount of toxigenic Pasteurella multocida antigenseffective to protect the mammal against a toxigenic Pasteurellamultocida infection.
 19. A method for protecting a pig against aBordetella bronchiseptica infection and/or a toxigenic Pasteurallamultocida infection comprising vaccinating the pig with an amount of avaccine according to claim 18 effective to protect against a Bordetellabronchiseptica infection and a toxigenic Pasteuralla multocidainfection.
 20. A method for protecting a piglet against atrophicrhinitis which comprises: a) vaccinating a pig prior to furrowing andduring pregnancy with an amount of a vaccine according to claim 18effective to provide anti-Bordetella bronchiseptica antibodies andanti-toxigenic Pasteurella multocida antibodies in the colostrumproduced by said female pig; and b) providing colostrum produced by saidfemale pig to the piglet within about 24 hours subsequent to thepiglet's birth.